Efficacy and safety of a novel hemostatic material, BoneStat, compared with Ostene and Bone Wax in a rat calvarial defect model.

Hemostasis has critical significance during surgical procedures. Bone Wax has traditionally been commonly used for bone hemostasis despite well-documented undesirable side effects: hindering osteogenesis and induction of inflammatory reactions with consequent increase in infection rates. A later developed formulation, Ostene, offers an alternative to Bone Wax with lesser undesired effects. In this study, BoneStat, a newly developed bone hemostatic formulation comprising water-soluble alkylene oxide co-polymers, was evaluated for water solubility, hemostatic efficacy, ease of handling, bone healing efficacy, and inflammatory reactions compared with Bone Wax and Ostene in a rat calvarial defect model. More than 95% of BoneStat was dissolved in water within 48 h, as was Ostene, but not Bone Wax. The time to hemostasis using BoneStat was significantly faster than with Ostene or Bone Wax. BoneStat also improved ease of handling compared to Ostene or BoneWax. BoneStat- and Ostene-treated groups constantly showed better bone healing than with Bone Wax. The BoneStat and Ostene groups presented no evidence of chronic inflammation reaction contrary to Bone Wax. These results suggest improved hemostasis, ease of handling, non-hindering bone healing, and unnoticeable chronic inflammatory reactions with BoneStat. Thus, Bonestat is a useful and reliable formulation for mechanical hemostasis.

Associations between biomarkers and histological assessment in individual animals in a destabilization of the medial meniscus (DMM) model of osteoarthritis (OA).

To date, the use of biomarkers for assessing individual severity of osteoarthritis (OA) is limited, and the correlation of histological scores with biomarkers for individual animals in the destabilization of the medial meniscus (DMM) model of OA has not been well investigated. Accordingly, this study investigated how well representative biomarkers in the DMM model reflected specific changes in individual animals. Rats were randomly divided into the OA group and the sham group. OA model was established by destabilization of the medial meniscus (DMM). After 2,4,6,8,10 and 12 weeks (n=14, each week), the concentrations of CTXII, COMP, C2C, and OC in serum were measured, and cartilage degeneration, osteophytes, and synovial membrane inflammation, typical of OA, were scored using Osteoarthritis Research Society International (OARSI) scoring system. Additionally, the correlation between each biomarker and the specific changes in osteoarthritis was analyzed for individual animals using the Generalized Estimating Equation (GEE). Statistical analysis showed a low correlation between CTXII and osteophyte score of the medial femur (coefficient = -0.0088, p= 0.0103), COMP and osteophyte score of the medial tibia (coefficient = -0.0911, p= 0.0003), and C2C and synovial membrane inflammation scores of the medial femoral (coefficient = 0.054, p= 0.0131). These results suggest that representative OA bio- markers in individual animals in the DMM model did not reflect histological scores well.

Comparative analysis of differentially secreted proteins in serum-free and serum-containing media by using BONCAT and pulsed SILAC.

Despite the increased interest in secretomes associated with paracrine/autocrine mechanisms, the majority of mass spectrometric cell secretome studies have been performed using serum-free medium (SFM). On the other hand, serum-containing medium (SCM) is not recommended very much because the secretome obtained with SCM is easily contaminated with fetal bovine serum (FBS) proteins. In this study, through the combination of bioorthogonal non-canonical amino acid tagging (BONCAT) and pulsed-SILAC (pSILAC), we analyzed differentially secreted proteins between SFM and SCM in a cancer-derived human cell, U87MG, and a mesenchymal stem cell derived from human Wharton's jelly (hWJ-MSCs). In most cases, the bioinformatic tools predicted a protein to be truly secretory when the secretion level of the protein was more in SCM than in SFM. In the case of hWJ-MSCs, the amount of proteins secreted in SCM for 24 hours was larger than that of SFM (log2 fold change = 0.96), even considering different cell proliferation rates. hWJ-MSCs proteins secreted more in SCM included several positive markers of MSC paracrine factors implicated in angiogenesis, neurogenesis and osteogenesis, and upstream regulators of cell proliferation. Our study suggests the analysis of the secretome should be processed in SCM that promotes cell proliferation and secretion.

Effect of Transplanting Various Concentrations of a Composite of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells and Hyaluronic Acid Hydrogel on Articular Cartilage Repair in a Rabbit Model.

BACKGROUND: Mesenchymal stem cells (MSCs) are known to have therapeutic potential for cartilage repair. However, the optimal concentration of MSCs for cartilage repair remains unclear. Therefore, we aimed to explore the feasibility of cartilage repair by human umbilical cord blood-derived MSCs (hUCB-MSCs) and to determine the optimal concentrations of the MSCs in a rabbit model. METHODS: Osteochondral defects were created in the trochlear groove of femur in 55 rabbits. Four experimental groups (11 rabbits/group) were treated by transplanting the composite of hUCB-MSCs and HA with various MSCs concentrations (0.1, 0.5, 1.0, and 1.5 x 107 cells/ml). One control group was left untreated. At 4, 8, and 16 weeks post-transplantation, the degree of cartilage repair was evaluated grossly and histologically. FINDINGS: Overall, transplanting hUCB-MSCs and HA hydrogel resulted in cartilage repair tissue with better quality than the control without transplantation (P = 0.015 in 0.1, P = 0.004 in 0.5, P = 0.004 in 1.0, P = 0.132 in 1.5 x 107 cells/ml). Interestingly, high cell concentration of hUCB-MSCs (1.5×107 cells/ml) was inferior to low cell concentrations (0.1, 0.5, and 1.0 x 107 cells/ml) in cartilage repair (P = 0.394,P = 0.041, P = 0.699, respectively). The 0.5 x 107 cells/ml group showed the highest cartilage repair score at 4, 8 and 16 weeks post transplantation, and followed by 0.1x107 cells/ml group or 1.0 x 107 cell/ml group. CONCLUSIONS: The results of this study suggest that transplantation of the composite of hUCB-MSCs and HA is beneficial for cartilage repair. In addition, this study shows that optimal MSC concentration needs to be determined for better cartilage repair.

Differential expression of extracellular matrix proteins in senescent and young human fibroblasts: a comparative proteomics and microarray study.

The extracellular matrix (ECM) provides an essential structural framework for cell attachment, proliferation, and differentiation, and undergoes progressive changes during senescence. To investigate changes in protein expression in the extracellular matrix between young and senescent fibroblasts, we compared proteomic data (LTQ-FT) with cDNA microarray results. The peptide counts from the proteomics analysis were used to evaluate the level of ECM protein expression by young cells and senescent cells, and ECM protein expression data were compared with the microarray data. After completing the comparative analysis, we grouped the genes into four categories. Class I included genes with increased expression levels in both analyses, while class IV contained genes with reduced expression in both analyses. Class II and Class III contained genes with an inconsistent expression pattern. Finally, we validated the comparative analysis results by examining the expression level of the specific gene from each category using Western blot analysis and semiquantitative RT-PCR. Our results demonstrate that comparative analysis can be used to identify differentially expressed genes.

Time-dependent differential gene expression in lysophosphatidic acid-treated young and senescent human diploid fibroblasts.

The gene expression profiles of lysophosphatidic acid (LPA)-treated young and senescent human diploid fibroblasts (HDFs) were examined using cDNA microarray analysis. The expression of some genes, including EGR 1/3 and MRRF, was controlled by LPA similarly in young and senescent cells, showing a typical time-dependent up-and-down expression profile. In contrast, some other genes, including DUSP6, CYR61, and F3, showed sustained upregulation in senescent HDFs later after LPA treatment. These genes might be involved in altered LPA responsiveness during the aging process.

Lysophosphatidic acid and adenylyl cyclase inhibitor increase proliferation of senescent human diploid fibroblasts by inhibiting adenosine monophosphate-activated protein kinase.

This study was designed to elucidate the molecular mechanism underlying lysophosphatidic acid (LPA) and adenylyl cyclase inhibitor SQ22536 (ACI)-induced senescent human diploid fibroblast (HDF) proliferation. Because adenosine monophosphate (AMP)-activated protein kinase (AMPK) is known to inhibit cell proliferation, we examined the phosphorylation status of AMPK and p53 and the expression level of p21(waf1/cip1) after treating HDFs with LPA and ACI. Phosphorylation of AMPKalpha on threonine-172 (p-Thr172-AMPKalpha) increases its catalytic activity but phosphorylation on serine-485/491 (p-Ser485/491-AMPKalpha) reduces the accessibility of the Thr172 phosphorylation site thereby inhibiting its catalytic activity. LPA increased p-Ser485/491-AMPKalpha, presumably by activating cAMP-dependent protein kinase (PKA). However, ACI reduced p-Thr172-AMPKalpha by inhibiting the LKB signaling. Our data demonstrated that both LPA and ACI inhibit the catalytic activity of AMPKalpha and p53 by differentially regulating phosphorylation of AMPKalpha, causing increased senescent cell proliferation. These findings suggest that the proliferation potential of senescent HDFs can be modulated through the regulation of the AMPK signaling pathway.

Role of protein kinase C-dependent A-kinase anchoring proteins in lysophosphatidic acid-induced cAMP signaling in human diploid fibroblasts.

Previously, we reported that lysophosphatidic acid (LPA)-induced adenosine 3',5'-cyclic monophosphate (cAMP) production by human diploid fibroblasts depends on the age of the fibroblasts. In this study, we examined the role of A-kinase anchoring proteins (AKAP) in the regulation of LPA-stimulated cAMP production in senescent fibroblasts. We found that levels of protein kinase C (PKC)-dependent AKAPs, such as Gravin and AKAP79, were elevated in senescent cells. Co-immunoprecipitation experiments revealed that Gravin and AKAP79 do not associate with adenylyl cyclase type 2 (AC2) but bind to AC4/6, which interacts with calcium-dependent PKCs alpha/beta both in young and senescent fibroblasts. When the expression of Gravin and AKAP79 was blocked by small interference RNA transfection, the basal level of cAMP was greatly reduced and the cAMP status after LPA treatment was also reversed. Protein kinase A showed a similar pattern in terms of its basal activity and LPA-dependent modulation. These data suggest that Gravin and to a lesser extent, AKAP79, may play important roles in maintaining the basal AC activity and in coupling the AC systems to inhibitory signals such as Gialpha in young cells, and to stimulatory signals such as PKCs in senescent cells. This study also demonstrates that Gravin is especially important for the long-term activation of PKC by LPA in senescent cells. We conclude that LPA-dependent increased level of cAMP in senescent human diploid fibroblasts is associated with increases in Gravin levels resulting in its increased binding with and activation of calcium-dependent PKC alpha/beta and AC4/6.

Lysophosphatidic acid-induced changes in cAMP profiles in young and senescent human fibroblasts as a clue to the ageing process.

This study attempts to elucidate the molecular mechanisms underlying the ageing-dependent cAMP profiles in human diploid fibroblasts stimulated by lysophosphatidic acid (LPA). In senescent cells, LPA-dependent Gialpha activation was reduced, with a consequent reduction in Gi-suppressed cAMP levels, without alterations in the levels of Gialpha proteins. In young cells, when Gialpha activity was inhibited by pertussis toxin pretreatment, or when its expression was blocked by siRNA, the pattern of changes in cAMP levels in response to LPA was similar to that seen in senescent cells. An increase in protein kinase C (PKC)-dependent isoforms of adenylyl cyclase (AC) types II, IV, and VI was also observed in these senescent fibroblasts. In senescent cells treated with PKC-specific inhibitors, bis-indolylmaleimide, Gö6976, rottlerin, and PKCvarepsilonV1, LPA-induced cAMP accumulation was inhibited, indicating that increased ACs in response to LPA occur via the activation of protein kinase Cs. When the expression of AC II, IV, and VI was blocked by siRNA in senescent fibroblasts, LPA-induced cAMP accumulation was also blocked. These results suggest that the senescence-associated increase of cAMP levels after LPA treatment is associated with reduced Gialpha, increased AC II, IV, and VI proteins, and PKC-dependent stimulation of their activities and provide an explanation for the age-dependent differences in cAMP-related physiological responses.